Statement of the Problem: TNF is a primary mediator of sepsis syndrome, systemic inflammation syndrome (SIS) and the adult respiratory distress syndrome (ARDS). TNF induces an increase in pulmonary vascular permeability in vivo, in the isolated lung and in pulmonary endothelial monolayers. There are many signal-pathways that are modulated in response to TNF such as different isoforms of protein kinase C (PKCs), myosin light chain kinase (MLCK), Mitogen Activated Protein Kinases (MAPKs), tyrosine kinases and capases of apoptosis. However, the role of these pathways in endothelial injury in response to TNF is still not very clear. Our studies indicate that PKCalpha, endothelial nitric oxide synthetase (eNOS) and NADPH-oxidase mediate the response to TNF; however, the paradigm that integrates PKCalpha, eNOS and NADPH-oxidase is not known. Our studies will show that a convergence pathway for PKCalpha , eNOS and NADPH-oxidase is peroxynitrite (ONOO_), formed by the interaction of nitric oxide (.NO) with superoxide (.O2), Our proposal will show that the endothelial barrier dysfunction in response to TNF is induced, at least in part, by the ONOO_ mediated nitration of critical tyrosine residues in cortical actin (i.e., nitrotyrosine-beta-actin). The nitrotyrosine-beta-actin polymers do not complex with actin-binding proteins of the zonular adherence (e.g., alpha-catenin) and/or zonular occludin (e.g., ZO-1) proteins, effectively enough, to maintain barrier function. The nitrotyrosine-beta-actin formation is because of an increase in ONOO( .NO+.O2 ->ONOO_) generated in the peripheral membrane. A novel mechanism for the increases in endothelial permeability in response to TNF will be investigated in this proposal. Hypothesis: The hypothesis of this proposal is that nitration of critical tyrosine residues in cortical-actin (i.e., nitrotyrosine-beta-actin) mediates, at least in part, the endothelial barrier dysfunction in response to TNF. Nitrotyrosine-beta-actin will not promote maintenance of F-actin polymers because of "capping-nitration" of the F-actin barbed ends, and the slow incorporation of the nitrotyrosine-beta-actin G-monomers into F-actin. Subsequently, the nitrotyrosine-beta-actin polymers do not complex with the actinbinding proteins of the zonular adherence and/or zonular occludin effectively enough to maintain barrier function. The nitrotyrosine-beta-actin formation is because of an increase in ONOO_ generation near the cortical beta-actin. The ONOO_ is generated from the reaction of .NO with .O2_ because of activation of NADPH-oxidase and eNOS, respectively, in the peripheral membrane. The activation of NADPH-oxidase is because of PKC" dependent phosphorylation of p47 phox. A novel mechanism for the increases in endothelial permeability in response to TNF will be investigated in this proposal. The Specific Objectives are: (1) To determine if PKCalpha-induced NADPH-oxidase activation mediates the ONOO_-induced change in nitrated-actin and actin morphology in pulmonary microvessel endothelial cell monolayers (PMEM) in response to TNF (Yearsl-2). (2) To determine which tyrosine residues of actin are nitrated that permit the change in actin morphology and increase in endothelial permeability in PMEM in response to TNF (Years 2-3) (3). To determine if nitrotyrosine-beta-actin mediates an increase in endothelial permeability because of a change in the incorporation of beta-actin with the actin-binding proteins of the zonular adherence and zonular occludin junctions, (-catenin and ZO-1, respectively (Years 3-4).